- Title
- Characterisation of and castanospermine effects on Foxp3+ and Foxp3- T cells in transplantation
- Creator
- LIU, Zilei
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Foxp3 positive CD4+ T cells (Tregs) play a crucial role in maintaining peripheral tolerance and promoting graft acceptance. The aim of this thesis was to characterize the Foxp3+ and Foxp3- T cells in the periphery before and after allo-activation and to assess a novel immunosuppressant, Castanospermine (CAST), on these cells and on the graft survival. The flow cytometric analysis of Foxp3+ and Foxp3+ cells in the normal (homeostatic) state in all the four T cell subsets, single CD4 positive (CD4+); single CD8 positive (CD8+); CD4/CD8 double positive (DP) and CD4/CD8 double negative (DN) showed that the vast majority of Foxp3+, CD25+, intracellular CTLA-4+, surface CTLA-4+, and PD-1+ T cells were limited to the CD4+ T cell subset whereas the highest expression level of CD25, intracellular CTLA-4, surface CTLA-4, PD-1 and Ki-67 was in DP T cells. The expression level and positive percentage of CD25, intracellular CTLA-4, surface CTLA-4, PD-1 and Ki-67 in Foxp3+ subpopulations were higher than Foxp3- subpopulations of the four T subsets, but none of these molecules were completely co-expressed with Foxp3 in any of the four T cell subsets. Although the expression of co-inhibitory molecules was higher on Tregs than on Tconv, Tregs maintained a much higher basal proliferation and had an even higher proliferation ratio after allo-activation than Tconv. Tregs and Tconv show different proliferation behaviour, the proliferation of Tregs was greater in vivo while poorer in vitro than Tconv. By employing mRFP reporter mice, in which the red fluorescent protein co-expresses with Foxp3, differential gene expression analysis between naïve Tregs (CD4+Foxp3+) and naïve Tconv (CD4+Foxp3-) was performed. Consistent with the flow cytometry data that Tregs maintained a much higher-level of Ki-67 than Tconv, the RNA differential expression analysis suggested that naïve Tregs exhibited “activated” signatures as they highly express genes encoding T cell activation markers such as CD25, CD69, and CD101, activation inducible co-stimulatory molecules such as ICOS and LAG-3 and major functional molecules in various intracellular signal transduction pathways. The differential gene expression pattern between naïve Tregs and naïve Tconv was similar to the differential gene expression between activated Tconv and naïve Tconv. However, this differential gene expression pattern was not shown in Tregs. CAST prolonged graft survival in this mouse skin graft model. The effects of CAST on the expression of functional molecules of Tregs were equivocal. For example, CAST slightly increased the expression of Foxp3 and CTLA-4 but suppressed the expression of CD25 and PD-1. Although CAST non-selectively suppressed the proliferation of Tregs, its inhibition of Tconv proliferation and partial blocking of inflammatory cells entering the grafts might be responsible for its prolongation of skin graft survival.
- Subject
- regulatory T cells; castanospermine; transplant tolerance; co-inhibitory molecules; Treg proliferation; differential gene expression
- Identifier
- http://hdl.handle.net/1959.13/1506853
- Identifier
- uon:55933
- Rights
- Copyright 2024 Zilei LIU
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 849 KB | Adobe Acrobat PDF | View Details Download |